Apparatus and method of making a gradient gel

ABSTRACT

An apparatus and method for making a gradient gel. The present invention in one embodiment is a gel-making system that has a reservoir for holding a solution. The reservoir is connected to a movable arm through a tubing. The tubing has two ends: one end is in fluid communication with the reservoir; and the other, an open end, is received by the movable arm. A gel holder having an internal gel chamber is placed underneath the movable arm for receiving the solution. In operation, the movement of the movable arm causes the open end of the tubing to move along with it and the open end of the tubing delivers the solution in motion to the internal gel chamber to form the gel.

[0001] This application is a divisional application of, and claimsbenefit of, U.S. application Ser. No. 09/219,402, filed Dec. 23, 1998which status is pending, the disclosure for which is hereby incorporatedherein in its entirety by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention comprises an apparatus and method formaking a gradient gel, and more particularly, the present inventionrelates to an apparatus and method of using a movable dispensing deviceto form a uniform linear gradient across a wide gel that provides morethan twenty sample lanes so that more than forty samples can be analyzedsimultaneously with a conventional dual-gel electrophoretic chamber.

[0004] 2. Background Art

[0005] Description of the Prior Art

[0006] Sixty to seventy five percent of the cholesterol in blood isassociated with low density lipoproteins (“LDL”) which consist of anon-homogeneous mixture of spherical particles ranging widely inparticle size (23-28 nm), buoyant density and chemical composition.Using a non-denaturing 2-16% polyacrylamide gradient gelelectrophoresis, researchers have noted that individuals with ahigh-risk lipid profile were most likely to have primarily small, denseLDL particles, as discussed in the paper “Genetic control of low densitylipoprotein subclasses,” Austin et al., Lancet 2: 592-595(3) (1986). Ina case-control study of men and women with documented myocardialinfarction (MI) published in the paper “Low-density lipoprotein subclasspatterns and risk of myocardial infarction,”Austin et al., J. Amer. Med.Assoc. 260: 1917-1921(4) (1988), it was reported that LDL phenotype B,the LDL subclass pattern characterized by a preponderance of small denseLDL particles, was associated with a 3-fold increased risk of MI. Thisassociation remained significant after adjustment for age, sex andrelative weight. It has also been suggested that there may be a majorgenetic determinant for this LDL phenotype as in the paper “Inheritanceof Low-density lipoprotein subclass patterns: results of complexsegregation analysis,” Austin et al., Am J Hum Genet 73: 838-876 (5)(1988). Whether or not the relationship between LDL phenotype and CAD isindependent of other risk factors such LDLc, HDLc or TRIG is stillunclear.

[0007] High density lipoproteins (HDL) are responsible for the reversetransport of cholesterol from peripheral tissues back to the liver. Datafrom the paper “Altered particle size distribution of apoA-I-containinglipoproteins in subjects with coronary artery disease,” Cheung et al.,J. Lipid Res. 32: 383-397 (1991), and the paper “Characterization ofhuman high density lipoproteins by gradient gel electrophoresis,”Johansson et al, Biochim Biophys Acta 665: 708-719 (1991), would suggestthat patients with documented CAD may have altered HDL particle sizedistribution when compared to that observed in non-CAD controls. Inthese studies, the heterogeneity of plasma HDL was assessed using anon-denaturing 4-30% polyacrylamide gradient gel first described in thepaper “Characterization of human high density lipoproteins by gradientgel electrophoresis,” Blanche et al., Biochim Biophys Acta 665: 708-719(1981).

[0008] A major impediment to large prospective studies of lipoproteinparticle size distribution has been the unavailability of an efficientand reproducible method that can allow the determination of particlediameters for cholesterol-rich lipoproteins. This is mainly because highquality pre-cast gradient gels used in the earlier studies are no longeravailable commercially. The paper “Production of polyacrylamide gradientgels for the electrophoretic resolution of lipoproteins,” Rainwater etal., J. Lipid. Res. 33: 1876-1881 (1992), has reported a procedure forthe preparation of a 4-30% gradient gel which provides estimates of HDLparticle size comparable to those obtained with the PAA 4/30 gel(Pharmacia). In this gradient, however, LDL and larger lipoproteinparticles tend to accumulate at the top of the gel, prohibiting thedetermination of particle size of these lipoproteins. A custom-made2-16% gradient gel was also described by these investigators for thedetermination of LDL particle size in the paper “Effects of diabetes onlipoprotein size,” Singh et al., Arterioscl. Thromb. Vasc. Biol. 15:1805-1811 (1995). Except for Gambert et al., who used lipid staining tovisualize the LDL band as disclosed in the paper “Human low densitylipoprotein fractions separated by gradient gel electrophoresis:Composition, distribution and alterations induced by cholesteryl estertransfer protein,” J. Lipid. Res. 31: 1199-1210 (1990), mostinvestigators used Coomassie to stain the gels for protein after theelectrophoresis. The use of a protein stain typically requires extensivestaining and de-staining procedures for the gels after electrophoresisand special handling of the gels during these steps to maintain gel sizeand shape before scanning. Furthermore, by using a protein stain, manyprotein bands other than those corresponding to plasma lipoproteins arevisible from the electrophoresis of whole plasma.

[0009] It is very difficult to make high quality of gradient gels formedical studies and clinic use. In the casting of the typical gradientgels, as shown in Rainwater et al. paper, the polyacrylamide solutionsare commonly allowed to flow into a gel chamber from a stationarydispensing tip which is typically placed at the center of the gel.However, as the polyacrylamide solution flows from the dispensing tip tothe sides of the plate, a secondary gradient is formed across the widthof the gel resulting in lower gel concentrations toward the edgesbecause of the diffusion of the solution. In order to reduce thisdiffusion effect, only narrow gels with 6-8 lanes across have beenavailable to-date although a typical gel chamber is capable of havinggels with up to 20 or more lanes. Moreover, uneven gradients anddisturbances in the process of gel making due to the diffusion stillexist even in the narrow gels.

SUMMARY OF THE INVENTION

[0010] Definitions

[0011] A number abbreviations used in this application for somefrequently used technical terms are defined as the following:

[0012] The term “S-GGE” as used herein shall refer to a segmentalgradient gel electrophoresis.

[0013] The term “S-GGE 2.8/8.30” as used herein shall refer to a2.8/8.30 segmental gradient gel electrophoresis with a 2-8% gradientstacked above an 8-30% gradient.

[0014] The term “LIPOPROTEIN” as used herein shall refer to a class ofplasma proteins that are complexed to lipids.

[0015] The term “TRIG” as used herein shall refer to triglycerides.

[0016] The term “CHOL” as used herein shall refer to cholesterol.

[0017] The term “LDL” as used herein shall refer to low densitylipoproteins.

[0018] The term “HDL” as used herein shall refer to high densitylipoproteins.

[0019] The term “Lp(a)” as used herein shall refer to lipoprotein(a)which consist of one LDL particle complexed to one apo(a) particle.

[0020] The term “LpB” as used herein shall refer to apoB-containinglipoproteins.

[0021] The term “LDLc” as used herein shall refer to LDL-cholesterol.

[0022] The term “HDLc” as used herein shall refer to HDL-cholesterol.

[0023] The term “LpA-I” as used herein shall refer to apoA-I containinglipoproteins.

[0024] The term “LpA-I/A-II” as used herein shall refer to lipoproteinscontaining both apoA-I and apoA-II.

[0025] Summary

[0026] The present invention provides a new apparatus and method formaking a uniform gel including a uniform, continuous gradient gel inmany lanes occupying up to the capacity of a gel chamber. Moreover, thepresent invention can be practiced to produce a segmental gradient gelthat would provide optimal conditions for the simultaneouscharacterization of LDL, Lp(a) and remnant lipoproteins (2-8% gradient)and HDL subclasses (8-30% gradient) from whole plasma. Additionally, thepresent invention allows the bands corresponding to all of the majorlipid-carrying particles to be visualized without any handling of thegel. The present invention can also be practiced to make severalgradient gels simultaneously. In sum, the present invention offers anew, better, and efficient gel making apparatus and method.

[0027] The present invention in one embodiment is a gel-making systemthat has a reservoir for holding a solution. The reservoir is connectedto a movable arm through a tubing. The tubing has two ends: one end isin fluid communication with the reservoir; and the other, an open end,is received by the movable arm. A gel holder having an internal gelchamber is placed underneath the movable arm for receiving the solution.In operation, the movement of the movable arm causes the open end of thetubing to move along with it and the open end of the tubing delivers thesolution in motion to the internal gel chamber to form the gel.

[0028] In order to make a gradient gel, normally two solutions withdifferent concentrations are used. Accordingly, one embodiment of thepresent invention employs a gradient maker that consists of a reservoirhaving a first container and a second container. The first containerholds a first solution and the second container holds a second solution.A channel connects the first container and the second container with anoutlet connected to the second container and in communication with thechannel so that a fluid of the first solution and the second solution isformed at the outlet. A tubing having a first end and a second endconnects to the outlet with the first end. The second end of the tubingis received by a movable arm and moves along with the movable arm. A gelholder with an internal gel chamber is placed underneath the movable armfor receiving the fluid, where the chamber has a longitudinal axis. Themovable arm moves back and forth along the longitudinal axis so that thesecond end of the tubing delivers the fluid in motion in the gel chamberto form the gradient gel. In a linear gradient gel the bottom of the gelhas a higher concentration and the top of the gel has a lowerconcentration.

[0029] A linear gradient gel can be formed from more than two solutions.In another embodiment of the present invention, a reservoir has aplurality of containers holding a plurality of solutions. Each containerholds one solution and communicates with at least one neighboringcontainer. An outlet is connected to at least one container tocommunicate with the containers so that a fluid of at least twosolutions from the plurality of solutions is formed at the outlet. Atubing, having a first end and a second end, is connected to (and is influid communication with) the outlet through the first end. A movablearm receives the second end of the tubing and causes the second end ofthe tubing to move along with it. A gel holder with an internal gelchamber is placed underneath the movable arm for receiving the fluid,where the chamber has a longitudinal axis. The movable arm moves backand forth along the longitudinal axis of the chamber so that the fluidis transferred from the first end to the second end of the tubing andthen is delivered in the chamber by the second end of the tubing inmotion to form the gradient gel.

[0030] In gel making, approximately two hours are required for the gelsolution to polymerize and form a solid matrix. One advantage of thepresent invention is that several highly uniform gradient gels can bemade simultaneously. In one embodiment of the present invention, aplurality of reservoirs are utilized. Each of them has a first containerand a second container, where the first container holds a first solutionand the second container holds a second solution. A channel connects thefirst container and the second container. Moreover, an outlet isconnected to the second container and communicates with the channel.Consequently, a fluid of the first solution and the second solution isformed at the outlet of this particular reservoir. Thus, a plurality offluids are formed at the plurality of the outlets of the plurality ofthe reservoirs. Furthermore, a plurality of tubings, each having a firstend and a second end, connect in a one to one relationship to theplurality of reservoirs through the connection of the second end of atubing to the outlet of a reservoir. A movable arm carries at least aplurality of the second ends of the plurality of the tubings. And aplurality of gel holders are positioned in parallel thereby defining alongitudinal axis. Each of the gel holders has an internal gel chamberfor receiving the fluid from one of the plurality of the second ends.When the movable arm moves back and forth along the longitudinal axis,each second end of the tubings delivers one of the fluids in motion intoone gel chamber to form one gradient gel. As a result, a plurality ofthe gradient gels are produced.

[0031] In order to make an uniform gradient gel, the movable arm movesat a substantially constant rate of motion. While other mechanisms maybe used, one embodiment of the present invention uses a motor to drivethe movable arm. One advantage of using the motor driving mechanism isthat by adjusting the speed of the motor, the rate of motion of themovable arm can be selected. In order to ensure that the same gelconcentration is present across the width of the gel, the rate of motionof the moveable arm is adjusted and set according to the width of thegel and the rate of flow of the solution from the reservoir into the gelchamber. For instance, for a wider gel, the rate of motion should beincreased to cover a greater distance in the same interval of time.Also, the height of the reservoir relative to the gel chamber can affectthe flow rate, which can be readily taken into account when setting therate of motion of the movable arm. According to the preferredembodiment, the movable arm has an internally threaded bore and themotor controls the motion of the movable arm through a shaft. The shafthas an elongated body with an external thread on the elongated body. Theshaft has a longitudinal axis and is rotatable around its longitudinalaxis. The shaft can rotate around the longitudinal axis either inclockwise direction or counterclockwise direction, and the direction ofrotation of the shaft is changeable from clockwise to counter clockwise,or vice versa. The external thread on the elongated body is adapted tomate with the movable arm through the internally threaded bore. Theshaft operatively connects to the motor. Thus, when the motor causes theshaft to rotate around its longitudinal axis, the movable arm movesalong the longitudinal axis because the mating mechanism of the externalthread of the shaft with the internally threaded bore of the movablearm. Because the shaft can rotate around the longitudinal axis either inclockwise direction or counter-clockwise direction, the movable arm isable to move along the longitudinal axis both forward and backward. Aswitching mechanism may be used to control the direction of the rotationof the shaft.

[0032] The movable arm in turn receives an open end of the tubing, whichconnects with the reservoir at the other end, the open end moving withthe movable arm. To do so, the movable arm has means for holding atleast a portion of the tubing proximate to the open end of the tubing sothat at least the open end of the tubing moves along with the movablearm. In one embodiment of the present invention, the holding means is anopening sized to allow the open end of the tubing to pass through buthold at least the portion of the tubing proximate to the open end of thetubing therein. The movable arm may have several openings at differentlocations to allow several gels to be made simultaneously, or to give auser freedom to set up the user's devices. The openings can also havedifferent sizes to accommodate tubings with different sizes.Alternatively, other holding means, including a clamping device normallyused in laboratories such as a clamp, can be used to associate thetubing with the moveable arm.

[0033] The tubing is made from a flexible material so that it can movealong with the movable arm easily without impeding the flow of the fluidwithin the tubing. Many materials can be used. In one embodiment of thepresent invention, tubings made from Manosie silicone rubber by VWRScientific Products Corporation, located at Willard, Ohio are used. Inuse, the tubing transfers fluid from the reservoir and delivers thefluid in motion at a substantially constant rate of flow. The tubing maydeliver the fluid through a dispensing tip. Or, the fluid can bedelivered simply through an open end of the tubing.

[0034] Solutions to make a linear gradient gel normally arepolyacrylamide solutions. These solutions can be identified as highconcentration polyacrylamide solution, medium concentrationpolyacrylamide solution and low concentration polyacrylamide solution.For a preferred embodiment of the present invention, a solutionaccording to a mixing ratio of a 2.3 g acrylamide and 0.1 g N,N-methylene-bis-acrylamide in 100 ml of borate buffer is regarded as alow concentration polyacrylamide solution (2.4%), a solution accordingto a mixing ratio of a 10.24 g acrylamide and 0.43 gN,N-methylenebis-acrylamide in 100 ml of borate buffer is regarded as amedium concentration polyacrylamide solution (10.67%), and a solutionaccording to a mixing ratio of a 38.4 g of acrylamide and 1.6 gN,N′-methylene-bis-acrylamide in 100 ml of borate buffer is regarded asa high concentration polyacrylamide solution (40%).

[0035] These solutions have been used successfully in the presentinvention to produce high quality segmental linear gradient gels,namely, a 2-8% continuous linear gradient gel and a 8-30% continuouslinear gradient gel. In doing so in one embodiment of the presentinvention, a commercial gradient maker with a container A and acontainer B, such as a Hoefer GS 100, purchased from Hoefer ScientificInstr., San Francisco, Calif. is loaded with solutions. Container A andcontainer B connect to each other through a channel. An outlet connectsto the container B and communicates with the channel so that a mixedfluid of the first solution and the second solution is formed at theoutlet. For this embodiment, the higher concentration of solution isalways in container B. To make a 8-30% linear gradient gel, a highconcentration of polyacrylamide is in container B and a mediumconcentration of polyacrylamide is in container A. A tubing transfersthe mixed fluid of the high concentration of polyacrylamide and themedium concentration of polyacrylamide from the outlet to a dispensingtip. The dispensing tip can be a separate device. Or an open end of thetubing can function as the dispensing tip. A movable arm receives thedispensing tip. A gel holder having an internal gel chamber with alongitudinal axis is placed underneath the dispensing tip. The motion ofthe movable arm along the longitudinal axis causes the dispensing tip tomove along with movable arm and the dispensing tip delivers the fluid inmotion in the gel chamber. Because the motion of the movable arm can becontrolled at a substantially constant rate, the fluid can be evenlydelivered throughout the gel chamber. Moreover, because the movable armis capable of traveling the length of the gel chamber, the solution isdelivered directly to the gel chamber “on-the-spot.” Thus, a secondarygradient resulting from the diffusion of the solution from thedispensing tip to the edge of the gel is not formed. After a propercuring period, a uniform, high quality 8-30% gradient gel is produced.Since in the segmental linear gradient gel a second gel (2-8% segment)must be poured on top of the first gel (8-30% segment), the volume ofthe solutions is controlled so that the 8-30% gradient gel occupies halfspace of the gel chamber. As people skilled in the art appreciate, theexact volumes to be placed in the reservoir can be calculated from thedimension of the gel chamber before hand. Furthermore, the entire gelmaking process according to the present invention can be automated byplacing the right volumes of the solutions in the reservoir. The gel ispoured to completion until the reservoir is emptied.

[0036] The containers A and B are subsequently filled with a mediumconcentration of polyacrylamide (in container B) and a low concentrationof polyacrylamide (in container A) to make a 2-8% gradient gel. Theabove process is then repeated. And a uniform 2-8% gradient gel isformed on top of the 8-30% gradient gel. As a result, a segmental lineargel consisting of two continuous linear gradients is formed, which canthen be used for the simultaneous determination of the diameters of LDLand HDL from whole plasma. The matching of the concentrations at theinterface of the two linear gradients in this embodiment provides acontinuous transition between the two linear gradients.

[0037] In a further embodiment of the present invention, two or moregradient makers, each with two containers A and B, can be used tosimultaneously make two or more gradient gels in two or more gelchambers. Therefore, practicing the present invention is economic andefficient, in addition to the advantages of making better continuousgradient gels.

[0038] In an additional further embodiment of the present invention,multiple linear gradients can be stacked on top of another to allowoptimal separation of the macromolecules of interest. For example, thepresent invention can be practiced to make a linear gradient gel withthree segments. Similarly, gels having other concentration gradients canalso be made.

[0039] Other objects, advantages and uses for the present invention willbe more clearly understood by reference to the remainder of thisdocument.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWINGS

[0040]FIG. 1 is a schematic view of a gel making system according to thepresent invention.

[0041]FIG. 2 is a schematic view of a first alternative embodiment ofthe gel making system of the present invention.

[0042]FIG. 3 is a schematic view of a second alternative embodiment ofthe gel making system of the present invention.

[0043]FIG. 4 is a partial side view of a gel making system of thepresent invention.

[0044]FIG. 5 is a partial top view of the gel making system shown inFIG. 4.

[0045]FIG. 6 is a perspective view of a movable arm according to apreferred form of the present invention.

[0046]FIG. 7 is a cross-sectional side view of the movable arm shown inFIG. 6.

[0047]FIG. 8 shows reproducibility of lipoprotein particle sizedeterminations: (A) an actual photograph of a gradient gel with the sameplasma sample (CHOL=279, TG=65, HDLc=65 and Lp(a)=175 mg/dL) beingapplied to 17 of the 21 available lanes. Aliquots of this plasma samplewere also applied to different lanes (x-axis) for 5 different gels(different symbols) over a two-week period; (B) the calculated particlediameters (nm) are plotted on the vertical axis as function of lanenumber (horizontal axis) for each gel and the different symbolsrepresent results from different gels. The mean diameter ( - ), 1 SDrange ( - - - ) and 2 SD range ( - - ) are presented for LDL and Lp(a)particle diameter; and (C) same as (B) but for HDL₂ and HDL₃.

[0048]FIG. 9 identifies conditions for optimal lipoprotein bands on theS-GGE 2.8/8.30 gel (‘S’ indicates the lane to which the Standard(calibrator) sample was applied): (A) by varying the incubation periodfor pre-staining of whole plasma from 120 min (Lane 1), 60 min (Lane 2),30 min (Lane 3) to 15 min (Lane 4) it is showed that there was no effecton the position of the Lp(a), LDL, HDL₂ and HDL₃ bands; (B) the positionof the LDL band was not affected by the concentration of LDLc in the 10μl of sample applied to the lane. A concentrated preparation of LDL(LDLc=270 mg/dL, Lane 5) isolated by ultracentrifugation was dialyzedagainst normal saline (d: 1.006 and 0.01% EDTA) and diluted with d:1.006 density solution containing 0.01% EDTA to various concentrationsof cholesterol ranging from 160 (Lane 1), 80 (Lane 2), 50 (Lane 3), to40 (Lane 4) mg/dL. All diluted samples were pre-stained by incubation atroom temperature for 2 hours; and (C) the application of comparableconcentrations of cholesterol in the form of VLDL resulted inconsiderably broader bands which are less intensely stained as comparedto LDL and Lp(a). VLDL was isolated by ultracentrifugation and appliedat cholesterol concentrations of 100 mg/dL (Lane 1, 530 mg/dL of TG), 50mg/dL (Lane 2, 265 mg/dL of TG) and 25 mg/dL (Lane 3, 133 mg/dL of TG).

[0049]FIG. 10 shows gel scans for whole plasma, isolated Lp(a) andisolated LDL, where Plasma from a healthy normal control woman(CHOL=279, TG=65, HDLc=65, and Lp(a)=175 mg/dL) was used for thisexperiment: (A) two sharp and distinct peaks can be visualizedcorresponding to lipoprotein particles with diameters of 23 nm orgreater. In view of the low plasma TG and high Lp(a) levels in thisindividual, we postulated that this peak of larger diameter correspondedto Lp(a); (B) By density gradient ultracentrifugation, a fractionenriched in Lp(a) was isolated and confirmed by ELISA. Uponelectrophoresis, this fraction exhibited a major band at the sameposition as the ‘designated Lp(a)’ when whole plasma was used. There wasa minor peak of small LDL which can be visualized. This would beconsistent with contamination by LDL of the Lp(a) fraction which wasisolated at a density higher than plasma LDL; and (C) LDL isolated inthe density range d: 1.020-1.063 from the same plasma exhibited a singlepeak corresponding to the major LDL peak seen with whole plasma.

[0050]FIG. 11 shows gel scans of whole plasma samples obtained atdifferent times following the consumption of a fat-containing meal: (A)analysis of fasting plasma from a female patient with documented CAD(Lp(a)=85, CHOL=175, TG=125, HDLc=79 mg/dL). In addition to the peakscorresponding to Lp(a) (Peak 2), LDL (Peak 3), HDL₂ (Peak 4), HDL₃ (Peak5) and albumin (Peak 6), this sample had a prominent shoulder (Peak 1)to the left of the Lp(a) peak suggestive of the presence of largerlipoproteins; (B) same as (A) but after 2 hours showing a well-definedpeak; (C) same as (A) but after 4 hours; (D) same as (A) but after 10hours, showing Peak 1 became more of a shoulder to the left of the Lp(a)peak as was the case with the fasting plasma sample. This broad peak isbelieved larger than Lp(a) represents TG-rich remnants. The diameter ofPeak 1 is estimated at 36.7 nm with a range of 32 to 39 nm in differentsamples.

[0051]FIG. 12 shows comparison of HDL particle size with the SFBR 3/31gel system: (A) displays a photograph of a gradient gel depicting thecalibrator (Lane 1), whole plasma (Lane 2), LpB (Lane 3), LpA-I (Lane 7)and LpA-I/A-II (Lane 5) for one subject. The band in Lane 7 identifiedin the size range of plasma LDL does not contain apoB as determined byELISA and can be shown by FPLC to contain phospholipids, cholesterylesters, cholesterol, apoE and apoC's (data not shown); (B) showscorrelation between HDL diameters determined by protein staining usingthe SBFR 3/31 gel from Alamo Gels Inc. and by lipid staining using theS-GGE 2.8/8.30 gel after the diameters of the HDL calibrators wereadjusted to be comparable with the values reported by Northwest LipidResearch Laboratory. The triangles (D) correspond to peaks identifiedfrom LpA-I fractions and the circles (∘) indicate peaks identified fromLpA-I/LpA-II fractions.

[0052]FIG. 13 shows the effect of sample storage on LDL particlediameter, where plasma samples from 51 subjects including normolipidemiccontrols and patients with various forms of dyslipidemia wereelectrophoresed fresh (within 7 hrs of sample collection) and afterstorage at −80° C. (3 to 12 months in cryovials without any additives):no statistically significant difference was found between the twoestimates by two-tailed paired t-test. The ratio of the two estimates ofLDL particle diameters (Fresh/Frozen) is plotted as a function of LDLparticle diameter determined from freshly isolated plasma. The meanratio ( - ), 1 SD ( - - - ) and 2 SD lines ( - - ) are also plotted. Thevariability in the ratio reflects the combined effect of sample storageand gel reproducibility.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention is more particularly described in thefollowing examples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. As used in the specification and in the claims, “a” can meanone or more, depending upon the context in which it is used. Thepreferred embodiment is now described with reference to the FIGS. 1-13,in which like numbers indicate like parts throughout the FIGS. 1-13.

Overview

[0054] Referring generally to FIGS. 1-7, the present invention comprisesa gel making system that utilizes a movable dispensing mechanism andmethod to make high quality gels, especially continuous gradient gels.The present invention allows the reproducible preparation of gradientgels which can be as wide as desired in order to accommodate any numberof samples simultaneously. One embodiment of the present inventionaccommodates up to 21 samples per gel, or 42 samples per run with adual-gel electrophoretic chamber, compared to several samples per gelusing currently available gel making devices. The present inventionallows the application of pre-stained plasma samples and the gel can bescanned for particle size immediately at the end of the electrophoreticprocedure. Elimination of the extensive staining and de-staining stepsafter electrophoresis should also minimize the need to handle the gelpreventing any artifact that these steps may introduce. Equallyimportant, the use of a lipid stain allows the specific visualization ofonly the lipoprotein fractions present in whole plasma. Thus, thepresent invention provides a new device and method to facilitate medicalresearch in the related fields.

[0055] Referring now to FIG. 1, the gel making system 100 of the presentinvention, according to one preferred embodiment, has a reservoir 10 forholding gel making solutions, a tubing 20, a movable arm 30, and a gelholder 50. The reservoir 10 has two containers 12, 14, each of them isused for holding one solution. As shown in FIG. 1, container 12 holdssolution A and container 14 holds solution B. The containers 12 and 14are connected by a channel 16 so that the containers can be in fluidcommunication to each other. Cross-sectionally, channel 16 can besquare, oval, circular or in other geometrical shapes. An optionalstopcock or valve 18 may be used to control the fluid communicationbetween the containers 12, 14. Moreover, as people skilled in the artknow, the ratio of the cross-sectional dimension of the channel 16 tothe cross-sectional dimension of an outgoing channel 15 determines thecharacteristic of the gradients: a linear gel is generated when they arethe same and a nonlinear gel results when they are not.

[0056] An outlet 19 is connected to the outgoing channel 15 andcommunicates with the channel 16 so that a mixture of the solution A andthe solution B is formed by the time the solutions reach the outlet. Thecross-sectional area of the channel 16 determines how fast solution A incontainer 12 mixes with solution B in container 14. The location of theoutlet 19 is important but variable. In the embodiment shown in FIG. 1,the outlet 19 is connected to the container 14. Alternatively, theoutlet 19 can be connected to the container 12. Or the outlet 19 may beconnected to both of the containers 12, 14, for example, through thechannel 16, at a location near the stopcock 18. The location of theoutlet 19 impacts the arrangement of solutions in the containers 12, 14.For the embodiment shown in FIG. 1, container 14 always holds a solutionwith a higher concentration of polyacrylamide.

[0057] Reservoir 10 can have alternative arrangements of containers.FIG. 2 shows one alternative to the embodiment of the reservoir 10 shownin FIG. 1. In FIG. 2, reservoir 10′ has three containers 12, 13 and 14,each of them for holding one solution. As shown in FIG. 2, container 12holds solution A, container 13 holds solution B and container 14 holdssolution C. The containers 12, 13 and 14 are connected by a channel 16and an outgoing channel 15 so that the containers can be in fluidcommunication to each other. Optional stopcocks or valves 17 may be usedto control the fluid communication among the containers 12, 13 and 14 sothat a variety of fluids, such as A+B, A+C, B+C, or A+B+C, can be made.

[0058] Multiple reservoirs 10 can also be used in the present inventionas shown in FIG. 3, where two reservoirs 10 are used to provide capacityfor making two gradient gels simultaneously as discussed in detail videinfra.

[0059] Still referring to FIG. 1, the tubing 20 has two ends 21, 23. Thetubing 20 is connected to the outlet 19 through the end 21 and is influid communication with the outlet 19. End 23 is an open end and isreceived by the movable arm 30.

[0060] The movable arm 30, referring to FIGS. 1, 6 and 7, has a body 32and openings 36 a, 36 b. Each opening is sized to allow the end 23 ofthe tubing to pass through and hold at least a portion of the tubing 20proximate the end 23 of the tubing therein. In the embodiment shown inFIG. 1, the tubing 23 is received by opening 36 a. Alternatively, it canbe received by opening 36 b. If multiple reservoirs are used as shown inFIG. 3, each opening would receive a tubing. The movable arm 30 can haveone, or two, or more openings to accommodate the need of a user.Moreover, each opening can be sized differently to accommodate the sizeof the tubing. Furthermore, cross-sectionally each opening may becircular, square, rectangular, triangular, diamond, oval or othergeometrical shape. Accordingly, openings can be cylinders (as shown inFIG. 7), cubic, cone or other geometric shapes. The movable arm 30 canalso take different shape. Cross-sectionally the body 32 may becircular, square, rectangular, triangular, diamond, oval or othergeometrical shape. Other alternatives can also be used to associate thetubing 20 with the movable arm 30. For example, a clamp (not shown) canbe used to associate the tubing 20 with the movable arm 30.

[0061] A gel holder 50 is placed underneath the end 23 and the movablearm 30. The gel holder 50 has an internal gel chamber 51 defined by twospacers 52, 54, two glass plates 56, 58, and a bottom 60. The chamber 51has an open top 53 to allow the fluid to be delivered therein throughthe end 23 of the tubing 20. The fluid can be delivered into the chamber51 directly through the open end 23 of the tubing 20. Optionally, adispensing tip (not shown) can be used to deliver the fluid.

[0062] The movable arm 30, in this embodiment, is driven by a motor 40.One advantage of using the motor 40 to drive the movable arm is that byadjusting the speed of the motor 40, the rate of motion of the movablearm can be selected. Once a rate of motion is decided, the motor 40 cankeep the movable arm 30 moving at that rate of motion constantly. A widerange of motors may be used. For the preferred embodiment of the presentinvention, motor 40 is an IG P/N 13556-165730 motor manufactured byIgurashi Electric Works, Japan.

[0063] The motor 40 controls the motion of the movable arm 30 through ashaft 70. According to the embodiment shown in FIGS. 4-6, the movablearm 30 has an internally threaded bore 34. The shaft 70 has an elongatedbody 72 with an external thread 74. The external thread 74 on theelongated body 72 is adapted to mate with the movable arm 30 through theinternally threaded bore 34. The shaft 70 has a longitudinal axis L andis rotatable around its longitudinal axis. The shaft 70 can rotate bothclockwise and counterclockwise. The direction of the rotation of theshaft 70 is controlled by switches 92 a, 92 b. Switches 92 a, 92 bchange the direction of rotation of the shaft 70 from clockwise tocounter-clockwise, or vice versa. Switches 92 a, 92 b may be pressuresensors, optical-electrical device, electromagnetic device or otherdevices. In this embodiment, switches 92 a, 92 b are double pole, doublethrow type 8221SHZGE switches, which can be found in most hardwarestores. The shaft 70 operatively connects to the motor 40. Thus, whenthe motor 40 causes the shaft 70 to rotate around its longitudinal axisL, the movable arm 30 is driven to move along the longitudinal axis Lbecause the mating mechanism of the external thread 74 of the shaft 70with the internal thread 35 of the internally threaded bore 34 of themovable arm 30. Because the shaft 70 rotates around the longitudinalaxis either in clockwise direction or counter-clockwise directionalternately, the movable arm 30 is able to move along the longitudinalaxis forward and backward continuously. Other mechanisms can be used tocontrol the motion of the movable arm 30. For example, an air gauge canbe used to drive the movable arm 30. Additionally, electromagneticmechanism and spring mechanism may also be utilized.

[0064] The movable arm 30, the motor 40 and the shaft 70 are supportedby a housing 80. Referring now to FIGS. 4 and 5, housing 80 has ahorizontal base 82 with corners 84 a-d. Two supporting posts 86 a, 86 bare bearing against the corners 84 a, 84 b. Crossing the horizontal base82, a supporting board 87 is bearing against the corners 84 c, 84 d.Alternatively, supporting board may be replaced by more supportingposts. Moreover, the positions of the supporting board 87 and thesupporting posts 86 a, 86 b are interchangeable. The supporting posts 86a, 86 b are connected by a horizontal bar 88 a, and the supporting board87 is connected to the supporting posts 86 a, 86 b by horizontal bars 88b, 88 c. In the embodiment shown in FIGS. 4 and 5, the horizontal bars88 b and 88 c are parallel to each other and therefore define alongitudinal axis. The supporting board 87 and the horizontal bar 88 aare parallel to each other but perpendicular to the longitudinal axis.Optionally, the horizontal bars 88 a-c are movably connected to thesupporting posts 86 a, 86 b and the supporting board 87 so that theheights of the horizontal bars 88 a-c relative to the horizontal baseare adjustable individually. The shaft 70 connects the horizontal bar 88a and the supporting board 87 and divides the housing 80 into twocompartments 90 a, 90 b. The motor 40 is operatively connected to theshaft 70 and also supported by the horizontal bar 88 a. The switches 92a, 92 b are mounted on the horizontal bar 88 a and the supporting board87 respectively.

[0065] Each of the compartments 90 a, 90 b can be used to receive a gelholder 50. Or two gel holders 50 can be received at once. Alternatively,by expanding the size of the housing 80 and length of the movable arm30, more gel holders can be placed within the housing 50 to make gels.Moreover, the height of the movable arm 30 relative to the horizontalbase 82 may be adjusted by adjusting the heights of the horizontal bars88 a-c to accommodate gel holders 50 with different heights.

[0066] Now the gel making system 100 is ready to be used. Referring nowto FIGS. 1, 4 and 5, the solution A from the container 12 and thesolution B from the container 14 form a mixture fluid at the outlet 19.The fluid travels through the tubing 20 to the open end 23. The motor 40is activated to drive the shaft 70 to rotate about the longitudinal axisL. The shaft 70 in turn, by the mating mechanism between the shaft 70and the movable arm 30, causes the movable arm 30 to move along thelongitudinal axis. When the movable arm reaches either switch 92 a orswitch 92 b, the rotational direction of the shaft 70 is changed fromclockwise to counter-clockwise, or vise versa. Because the change of thedirection of rotation of the shaft 70, the movable arm reverses it'sdirection of motion along the longitudinal axis as well. As a result,the movable arm moves back and forth between the horizontal bar 88 a andthe supporting board 87. In a continuous motion, the movable arm travelsback and forth along the longitudinal axis and causes the open end 23 ofthe tubing 20 to move along with it. During this oscillating motion, theopen end 23 delivers the fluid into the internal gel chamber 51 throughthe open top 53 between the two spacers 52 and 54. Because the movablearm 30 moves at a constant rate of motion, open end 23 delivers thefluid in motion at a substantially constant rate of flow. After a properamount of the fluid is deposited in the gel chamber 51, it can be curedto form the desired continuous gradient gel.

[0067] It is possible to make a second gradient gel on top of the newlymade gradient gel so that the two gels form a continuous concentrationchange from the bottom of the first gradient gel to the top of thesecond gradient gel. To do so, the volumes of the solutions A and B areselected so that the gradient gel is formed at the bottom half of thegel chamber 51. Then the containers 12, 14 are emptied and refilled withtwo new solutions and the above process is repeated to form a second gelon the top half of the gel chamber 51. Because the fluids are deliveredat substantially constant rate of motion and from side-to-side, theupper edge of the first gradient gel is rather smooth so as to supportthe second gel on the top half. Because the diffusion effect isminimized in the present invention, much wider and better quality gelsare produced according to the present invention.

[0068] While it is desirable to make a gel up to the capacity of the gelchamber by delivering the fluid in side-to-side motions, an optionalstopper can be put on the track of the movable arm 30 to customize thesize of the gel to be made. Furthermore, referring to FIGS. 3-5, two ormore gel holders can be placed in the housing 80 to simultaneously makemultiple gels. The solutions at each reservoir can be same or different,depending on a user's need.

[0069] The invention will be better understood by reference to thefollowing illustrative example, which is performed according to thepresent invention.

EXAMPLES

[0070] Materials

[0071] Polyacrylamide was obtained from Sigma Chemicals (A-3553 andM-7279), as were the ammonium persulfate (A-1433) and TEMED (T-9281) andthe stain, Sudan Black B (Sigma: S-0395). Ethylene glycol (E178-1) andboric acid (BP 168-500) were purchased from Fisher Scientifics.Tris(hydroxymethyl)aminomethane (EK-1 174952) was from VWR Scientifics.

[0072] Equipment

[0073] Some components of the gel apparatus as shown in FIGS. 1-7 werepurchased from Hoefer Scientific Instruments at San Francisco, Calif.and included the SE 650 vertical slab unit, the SG-100 gradient maker,and the PS-1500 power supply. The gradient gel was prepared using the18×8 slab gel unit from Hoefer (SE 6402) with a 3-mm spacer. Two 12-slotsample applicators from Isolabs (GC-50) were adapted for our gel systemto optimize sample loading. The gel was scanned using the LKB 222-020UltroScan XL Laser Densitometer. The oscillating motion of thedispensing arm was controlled by a platform mixer (Vari-Mix,Thermolyne). The movable arm and mating shaft were manufactured in theinventors' laboratory.

[0074] Buffers

[0075] Three stock preparations containing high, intermediate and lowconcentrations of polyacrylamide were available: (1) HIGH: 38.4 grams ofacrylamide and 1.6 g N,N′-methylene-bis-acrylamide in 100 ml of boratebuffer (8 mM boric acid, 90 mM Tris, 3 mM EDTA, pH 8.3) and (2) MEDIUM:10.24 g acrylamide and 0.43 g N,N′-methylenebis-acrylamide in 100 ml ofborate buffer and (3) LOW: 2.3 g acrylamide and 0.19 gN,N′-methylene-bis-acrylamide in 100 ml of borate buffer. A stocksolution of TEMED was prepared by combining 0.6 ml of TEMED and 99.4 mlof the borate buffer. Ammonium persulfate solution (5 mg/ml) was alsoprepared with the borate buffer.

[0076] Preparation of Segmental Gradient Polyacrylamide Gels (S-GGE2.8/8.30)

[0077] The segmental polyacrylamide gradient gel (3 mm thick) was pouredin two steps using the Hoefer SE 6402 gel maker kit with 18×8 cm glassplates and the gradient maker (Hoefer GS 100). Each container of thegradient maker was filled with a mixture of 6 ml of the appropriatepolyacrylamide concentration, 1 ml of TEMED buffer and 1 ml of ammoniumpersulfate solution. For the first stage, the two containers heldcontained the high (Container B) and medium (Container A) concentrationsof polyacrylamide to generate the 8-30% gradient. As shown in FIG. 1,the polyacrylamide solutions were allowed to flow continuously as themovable arm was moved from side to side along the upper rim of the glassplates, thus creating multiple tracks along the glass plates across thewidth of the gel chamber. This motion (15 side-to-side oscillations/min)ensured the even distribution of the polyacrylamide solution between theplates over the entire width of the gel (14 cm). The containers wereallowed to empty completely creating the lower gradient gel(approximately 3.8-4 cm in height) which was then allowed to polymerizeat room temperature (2 hours). The containers of the gradient makerswere subsequently filled with the medium (container B, 6 ml) and low(container A, 6 ml) concentration polyacrylamide to form the 2-8% lineargradient gel. To each container, 1 ml of TEMED buffer and 1 ml ofammonium persulfate solution were added for a total of 16 ml for bothchambers. The quality and reproducibility of the gel is optimal if therate of gel flow is matched to the appropriate rate of movement of thedispensing arm. The rate of flow is controlled by the mating mechanismbetween the shaft 70 and the movable arm 30 movable with a mean rate of1.5 ml/min and the moving arm moved at a rate of 15 cycles/min.

[0078] Electrophoresis

[0079] Whole plasma (30 μl) was mixed with 10 μl of a prestainingsolution (0.6% Sudan Black B solution in ethylene glycol) prior toapplication to the gel. After a 2-hour incubation period at roomtemperature, a single load of 10 μl of prestained plasma solution wasapplied to individual troughs, characteristic of the GA-50 sampleapplicator (Isolabs). Electrophoresis was performed at room temperature,50 mA, 80 V for 18-20 hours. Alternatively, the gel image can bedigitized using the Image Master Video Documentation System and AnalysisSoftware, which is commercially available.

[0080] Calibration of Lipoprotein Particle Size

[0081] Whole plasma from a human donor exhibiting distinct lipoproteinbands corresponding to LDL, Lp(a), HDL₂ and HDL₃ (CHOL=297, TG=97,HDLc=73 and Lp(a)=29 mg/dL) was run in multiple lanes of each gel. Thediameter of the LDL band in this plasma sample was calibrated using aset of in-house LDL calibrators (27.7, 25.3 and 24.2 nm). The diametersfor the in-house LDL calibrators had been previously standardizedagainst the calibrator pool ILH containing LDL with diameter of 29.7,27.1 and 24.7 nm available from Dr. Krauss (Donner Laboratory) aspreviously reported (16,17) using PAA 2/16 gels from Isolabs.

[0082] As with other gradient gel systems, the diameters of HDL₂ andHDL₃ in the S-GGE 2.8/8.30 gradient gel were determined using the highmolecular weight calibrators obtained from Pharmacia (HMW 17-0445-01).For these calibration runs, the gels were stained for protein withCoomassie after electrophoresis to visualize the protein bandscorresponding to the molecular weight standards. The high molecularweight calibrators included: thyroglobulin (17.0 nm), ferritin (12.2nm), catalase (10.4 nm), lactate dehydrogenase (8.4 nm), and albumin(7.1 nm). Frozen aliquots of plasma from this donor were maintained at−80° C. (up to 2 years) and included in all subsequent runs ascalibrators for the gel. The quality of the calibrators was assessed bythe actual position of the bands as well as the values calculated forthe gel constant. These aliquots were thawed once for each use anddiscarded without being re-frozen for later use.

[0083] Determination of Particle Size using the Gel Constant

[0084] From gel chromatography experiments, it is known that the poresize (S_(pore)) of polyacrylamide gel varied inversely to the monomerconcentration (T_(gradient)) i.e.

S _(pore) =K ₁[1/T_(gradent])  [Eq. 1]

[0085] where K₁ denotes the unknown proportionality constant. For alinear gradient, the monomer concentration T_(gradient) of thepolyacrylamide at any distance d in the gel is a function of thedistance d from the top of the gel, thus

T_(gradient) =f(d).  [Eq. 2]

[0086] The function f will also depend on the range and slope of thegradient. By combining Equations 1 and 2, we obtained:

S _(pore) =K ₁×[1/f(d)]  [Eq. 3]

[0087] When gradient electrophoresis is carried out to completion,spherical particles of uniform size will continue to migrate until theyreach a distance in the gradient, d_(particle), where the pore size ofthe gel matrix becomes so small as to prevent further penetration by theparticle. At this point of equilibrium the pore size is approximatelyequal to the particle size. In other words,

S _(particle) =S _(pore) =K ₁×[1/f(d _(particle))]  [Eq. 4]

or

C _(gel) =S _(particle) ×f(d _(particle))=S _(pore) ×f(d_(particle))  [Eq. 5]

[0088] By using a calibrator of known particle diameter S_(calibrator)and by measuring the distance d_(calibrator) migrated into the gel ofthis calibrator, we can calculate the gel constant, C_(gel), for thisparticular linear gradient.

C _(gel) =S _(calibrator) ×f(d _(calibrator))

[0089] Using the distances determined by the gel scanner for the bandscorresponding to the LDL, Lp(a), HDL₂ and HDL₃ calibrators, we cancalculate the gel constants for the upper and lower gels. Since thedistance d_(calibrator) depends ultimately on the polyacrylamideconcentration in the gel gradient, Eq. 5 would indicate that C_(gel)will depend only on the pore size and should, in theory, be the sameindependent of the gradient:

C _(gel)(nm-%)=S _(calibrator) ×[L+(d_(calibrator))*(Slope_(gradient))]  [Eq. 6]

[0090] where S_(calibrator) is the known diameter (nm) of the calibratorL is the lowest gel concentration for the gradient, i.e. 2% for the 2-8%gradient and 8% for the 8-30% gradient

[0091] d_(calibrator) (mm) is the position of the band corresponding tothe calibrator from the top of the gradient

[0092] Slope_(gradient) is the slope (% per mm) of the gradient, or thedifference between the lowest and the highest gel concentrations (6% forthe top gradient and 22% for the bottom gradient) divided by the heightof the gel. The actual height of each gradient can be determined by theoperator for each gel using the scanner.

[0093] 2-8% gradient:

[0094] with Lp(a) C_(gel)=30.40×[2+(9.8)*(6/35)]=111.863

[0095] with LDL C_(gel)=25.70×[2+(13.7)*(6/35)]=111.748

[0096] 8-30% gradient

[0097] with HDL₂ C_(gel)=11.80×[8+(38.40−35.0)*(22/37)]=118.255

[0098] with HDL₃ C_(gel)=9.60×[8+(41.70−35.0)*(22/37)]=115.044

[0099] From this estimate of the gel constant, the diameterS_(unknown of any particle can be calculated from the distance d)_(unknown) from the top of the gel. It should be noted that thedensitometer gives the position of the band as referenced to anarbitrary set point (typically, from the edge of the glass plate) andthe distance to the top of the gradient must be subtracted to obtain thetrue distance of migration into the gel. Furthermore, for the lowergradient gel, the actual height of the upper gel must also besubtracted:

S _(unknown) =C _(gel) /f(d _(unknown))

[0100] With each gel, the calibrator plasma containing Lp(a), LDL, HDL₂and HDL₃ of known particle diameters was always applied in 3 separatelanes including the two outside lanes and one toward the center of thegel. From the previously determined diameters of Lp(a) and LDL in thecalibrator plasma, 6 separate estimates (2 calibrators×3 lanes) wereobtained for the gel constant of the 2-8% gradient. The mean value wasused to determine the diameters of LDL and Lp(a) in the unknown samples.Similarly, the diameters of HDL₂ and HDL₃ in the calibrator plasmaallowed the calculation of 6 estimates for the gel constantcorresponding to the 8-30% gel gradient and the mean value was used incalculating the HDL particle diameter.

[0101] Lipoprotein Isolation

[0102] To examine the characteristics the different class oflipoproteins in this system, individual fractions were isolated byultracentrifugation from freshly collected plasma. VLDL was recovered inthe supernate of the 1.006 spin (24-hr spin at 39,000 RPM and 10° C.)using the SW 40 Swinging bucket rotor (Beckman Instruments, Palo Alto,Calif.). LDL was recovered in the density range of 1.019-1.063 gm/ml bysequential ultracentrifugation. Lp(a) was isolated by densityultracentrifugation using a modification of the method reported in thepaper “Physico-chemical properties of apolipoprotein(a) andlipoprotein(a-) derived from the dissociation of human lipoprotein(a),”Fless et al., J. Biol. Chem. 261: 8712-8718 (1986). In brief, 1 ml ofplasma was adjusted to a density of 1.050 gm/ml using solid KBr andlayered with 12 ml of a 1.040 density solution. Ultracentrifugation wasperformed at 35,000 RPM for 15 hrs (10° C.) using the SW 40 swingingbucket rotor. The fraction corresponding to Lp(a) was removed by carefulaspiration and confirmed by ELISA.

[0103] In order to compare our estimates obtained for HDL-sizedparticles with this new procedure, we used LpA-I and LpA-II/A-IIfractions which had been isolated by immunoaffinity chromatography asdiscussed in the paper “Altered particle size distribution ofapoA-I-containing lipoproteins in subjects with coronary arterydisease,” Cheung et al., J. Lipid. Res. 32: 383-397 (1991) andgenerously donated by Dr. Marian Cheung of the Northwest Lipid ResearchLaboratory. The diameters of the major protein fractions in these HDLsubclasses had been determined using the 4-30% gel available from AlamoGels, Inc (San Antonio, Tex.). By using these purified HDL subfractionsto compare the estimates of particle diameters obtained by the two gelswe can be sure that only peaks corresponding to HDL proteins arevisualized after staining with Coomassie.

Results

[0104] Reproducibility of the S-GGE 2.8/8.30 for Lipoprotein ParticleDiameter

[0105]FIG. 8A illustrates the reproducibility of the linear gradientacross the 21 lanes of the gel as demonstrated by identical distances ofmigration across all lanes for all four major lipoprotein bands. FIG. 8Bpresents the actual particle diameters for obtained for LDL and Lp(a)when the calibrator plasma was applied in multiple lanes of 5 differentgels. Only 9.4% (3/32) of the individual particle diameter estimates forLDL were outside 1 SD of the mean and none were outside 2 SD. For Lp(a),12.5% (4 out of 32 lanes) of the particle diameter estimates from 5separate gels were outside 1 SD of the mean. None were outside 2 SD. Forthe HDL subfractions, 18.8% (6 out of 32 lanes) were outside 1 SD and6.2% (2 out of 32) were outside 2 SD (FIG. 2C). Table 1 presents themean values and fractional standard deviation (100×SD/Mean) for thedistance from the top and for the calculated particle diameter forLp(a), LDL, HDL₂ and HDL₃ from 16 lanes on the same gel (within run) aswell as values from 65 different gels (between runs).

[0106] The reproducibility of the gel constants (mean±SEM for the mostrecent series of 100 gels) was obtained for both the 2-8%(C_(gel)=113.5±0.45) and the 8-30% linear gradients(C_(gel)=116.43±0.28). There was no statistical difference between thetwo estimates for the C_(gel) from the lower and upper gradient gels asassessed by un-paired t-test. These empirical results further supportthe earlier observation that the pore size at any point in a lineargradient is defined solely by the gel concentration, independent of therange of the gradient prepared.

[0107] In order to examine the effect of the pre-staining procedure onthe electrophoretic mobility of the lipoproteins, whole plasma aliquotsfrom a single donor were incubated in the staining solution for 15 min,30 min, 1 hr and 2 hrs at room temperature and electrophoresed inadjacent lanes. As shown in FIG. 9A, there was no change in the positionof any bands corresponding to the 4 major lipoprotein fractions. Table 2presents the actual particle diameters for 6 separate incubations at 15min, 1 hour and 2 hour for a freshly collected plasma sample whichdisplays all 4 major lipoprotein bands. There was no statisticaldifference in particle size with the different incubation times.

[0108]FIG. 9B illustrates the reproducibility of the position of LDLbands as different concentrations of LDLc were applied. With the presentprotocol for pre-staining and electrophoresis, particle sizedetermination for LDL can be determined from a sample with an LDLcconcentration of 40 mg/dL. While there appeared to be a dose-dependencebetween LDLc and the intensity of the stained LDL bands, we were notable to establish a reliable standard curve between the areas under theLDL peaks as determined from the densitometer and the concentrations ofLDL when plasma samples from different donors were analyzed. This is incontrast to the reported data obtained with protein staining.Differences in the lipid composition of LDL among individuals couldaccount for the variability in the stain intensity for differentpreparations of LDL.

[0109] In contrast to the sharp bands characteristic of LDL, small VLDLcan be shown to have significantly broader peaks which are poorlystained (FIG. 9C). For this experiment, VLDL from a normotriglyceridemicdonor (CHOL=195, TG=80, HDLc=56 and Lp(a)<0.1 mg/dL) was isolated byultracentrifugation using the SW40 swinging bucket at density d<1.006gm/ml. In contrast to the band corresponding to 40 mg/dL of LDLc ( Lane4, FIG. 9B), the band corresponding to 50 mg/dL of VLDL-cholesterol(Lane 3, FIG. 9C) was barely visible under identical stainingconditions. As shown, the band corresponding to VLDL from thisnormotriglyceridemic individual was slightly larger than Lp(a) in ourcalibrator (Lane S) and was significantly more heterogeneous asindicated by the broadness of stained band. In our hands, VLDL isolatedby ultracentrifugation of whole plasma obtained from individuals withfasting TG of 350 mg/dL or greater can be visualized as a dark stain atthe top of 2% gel suggesting that these particles were too large toenter the gel matrix. Using the value of gel constant calculated for the2-8% gel and Eq. 6, we would predict that only particles with diameterless than 55 nm would be able to enter the gradient.

[0110] Identification of Lp(a)

[0111] The identity of the Lp(a) band was confirmed by severalapproaches. First, the stain characteristics of the Lp(a) band aredifferent from that of VLDL (FIG. 9C). The Lp(a) band is sharper andmore comparable to that of LDL than that of VLDL. Secondly, isolatedfractions of Lp(a) can be shown to correspond to distinct peaks uponelectrophoresis in the S-GGE 2.8/8.30 gel with migration distanceidentical to the corresponding peaks obtained with whole plasma (FIG.10). And thirdly, analysis of postprandial plasma samples from anindividual demonstrated that the peak corresponding to Lp(a) wasunchanged while the area under the peak corresponding to remnantsincreased at 2 hours postprandially before returning to fasting levelafter 10 hours (FIG. 11).

[0112]FIG. 10A illustrates actual gel scans for a plasma sample with ahigh concentration of Lp(a). In FIG. 10B, we present the gel scan of apartially purified Lp(a) isolated by density gradientultracentrifugation using the SW40 swinging bucket as previouslydescribed by Fless et al. paper. The position of the Lp(a) peak was28.20 mm in whole plasma as compared to 28.52 mm for the isolated Lp(a)from the edge of the plate. This corresponds to a distance of 8.2-8.5 mmfrom the top of the gel gradient. The gel scan in FIG. 10C illustrates asingle peak for LDL isolated by ultracentrifugation in the density range1.020-1.063 g/ml from the same plasma. In all samples with Lp(a)concentrations of 35 mg/dL or greater as determined by ELISA, we haveconsistently been able to visualize a band at approximately 8.2-8.75 mmfrom the top of the gel corresponding to a particle diameter in therange of 27 to 30 nm.

[0113] To further demonstrate the ability of the gel to resolve smallVLDL and remnants from Lp(a), we examined non-fasting plasma from anindividual with a distinct Lp(a) band present in fasting plasma (FIG.11A). As shown in FIG. 11A, a distinct peak corresponding to Lp(a) wasnoted that was slightly larger than LDL and a subpopulation of evenlarger particles appeared as a shoulder (Peak 1) to the left of theLp(a) peak. Peak 1 was not visible in the gel scans from the plasma ofmost individuals with normal TG levels (<100 mg/dL, FIG. 10).Postprandial plasma samples collected following the consumption of astandardized liquid test meal containing fat and cholesterol (19,20)were subjected to electrophoresis (FIGS. 11(B)-(D)). At 2 and 4 hoursfollowing the test meal, when postprandial plasma TG were expected toincrease, this shoulder associated with larger lipoprotein particlesclearly became a distinct peak (Peak 1) with increasing areas (FIG. 11Band C). The position of this band, i.e. the diameter of this lipoproteinfraction, is not changed in postprandial plasma. By 10 hour after thetest meal, as plasma TG returned to fasting level, this band of largerlipoprotein particles was reduced back to a shoulder to the left of theLp(a) peak (FIG. 11D). The particle diameter corresponding to this peakis estimated to be 36.7 nm. We postulate that this peak of largerlipoproteins corresponded to TG-rich lipoproteins and their remnantswhich were generated during postprandial lipemia. This shoulder isdemonstrable in only a subset of the individuals studied with the oralfat load and its presence does not appear to be associated with fastingTG levels. Additional experiments are ongoing to further characterizethe nature of this lipoprotein peak. It is clear, however, that thesharp band in the size range from 27 to 30 nm must correspond to Lp(a)and not to IDL or other TG-rich remnant lipoproteins which wouldtypically have larger diameters ranging from 33.5 to 39 nm.

[0114] HDL Particle Size: Comparison with SFBR 3/31 Gel

[0115] In order to compare the diameters of HDL particles obtained withour gel system and the conventional Pharmacia PAA 4/30 gel, we examinedLpA-I and LpA-I/A-II fractions isolated by immunoaffinitychromatography. As mentioned above, these lipoproteins fractions werekindly provided by Dr. Marian Cheung Northwest Lipid ResearchLaboratory, University of Seattle, Seattle, Wash.) and the correspondingdiameters were determined using the 3-31% gels (SFBR 3/31, Alamo Gels,Inc., San Antonio, Tex.) following protein staining. FIG. 12Aillustrates the lipid-stained bands obtained for whole plasma, LpB,LpA-I and LpA-I/A-II for one individual. In contrast to theprotein-stained gels disclosed, for examples, in the paper “Alteredparticle size distribution of apoA-I-containing lipoproteins in subjectswith coronary artery disease,” Cheung et al., J. Lipid. Res. 32: 383-397(1991) and the paper “High density lipoproteins and coronaryatherosclerosis: A strong inverse relation with the largest particles isconfined to normotriglyceridemic patients,” Johansson et al., ArteriosclThromb 11: 177-182 (1991), only one or two major bands could bevisualized with the lipid stain. For most samples, two lipid-rich bandscan always be identified for LpA-I and only one for LpA-I/A-II. Only themajor bands with the greatest area under the peak based on the proteinstain were selected in this comparison. The two estimates of HDLparticle diameters for LpA-I and LpA-I/A-II from 11 individuals werehighly correlated (r=0.978 for a total of 37 peaks). We used this linearregression equation to calculate the adjusted particle diameters of theHDL₂ and HDL₃ in our calibrator. A new gel constant was derived usingthese adjusted diameters and FIG. 12B illustrated the correlationbetween the particle diameters for HDL subfractions determined on theS-GGE 2.8/8.30 using the gel constant approach and the values obtainedby Dr. Cheung using the SFBR 3/31 gel and the Rf approach. The slope ofthe linear regression was 0.948 with an intercept of 0.4 nm (r=0.97).

[0116] Effect of Sample Storage on LDL and Lp(a) Particle Diameter

[0117]FIG. 13 illustrates the reproducibility of lipoprotein particlesize between fresh samples and frozen samples from a group of 52individuals including normolipidemic controls and subjects with varyingdegrees of hyperlipidemia. Fresh plasma samples were analyzed as theywere available (within 7 hrs of collection) using as many as 20 separategels. Frozen samples were analyzed at the end of an 8-month storageperiod using four separate gels within a 2-week period. The mean (±SD)LDL particle diameter for the group was 25.7 nm (±0.807) based on theanalysis of freshly isolated plasma and 25.3 nm (±0.693) when analyzedfrom frozen plasma samples. There was no statistically significantdifference between the two measurements by two-tailed paired t-test. Themean (±SD) ratio of LDL diameter between the fresh and the frozenestimate was 1.0077 (±0.028). In 26.9% (17 of 51) of the samples, theratio of the two estimates of LDL particle diameters (fresh vs. frozen)was greater than 3.27% (outside 1 SD) following a single freeze-thawcycle. For 7.7% of the plasma samples (7 of 52), the ratio of the sizeestimates for LDL was greater than 6% (outside 2 SD) following a singlefreeze-thaw cycle. One third of the samples (16 of 52) were actuallystored for 12 months prior to reanalysis. Since there were nodifferences in the estimates for particle diameter with longer storage,results from these samples were included in the final analysis.

[0118] In the present report we have described a protocol for thepreparation of a gradient gel system consisting of two linear gradients,an 8-30% gradient for particles in the range of HDL and a 2-8% gradientfor LDL and larger lipoprotein particles. The present system offersseveral advantages over existing systems previously described in theliterature. Using commonly available gel casting equipment andconventional electrophoretic supplies, this protocol allows thereproducible preparation of gradient gels which can accommodate up to 21samples per gel, or 42 samples per run with a dual-gel electrophoreticchamber. The protocol allows the application of pre-stained plasmasamples and the gel can be scanned for particle size immediately at theend of the electrophoretic procedure. Elimination of the extensivestaining and de-staining steps after electrophoresis should alsominimize the need to handle the gel preventing any artifact that thesesteps may introduce. More importantly, the use of a lipid stain allowsthe specific visualization of only the lipoprotein fractions present inwhole plasma.

[0119] Although the present invention has been described with referenceto specific details of certain embodiments thereof, it is not intendedthat such details should be regarded as limitations upon the scope ofthe invention except as and to the extent that they are included in theaccompanying claims. Many modifications and variations are possible inlight of the above disclosure.

[0120] For example, a computer can be used to coordinate the motion ofthe movable arm and the rate of flow according to the width of the gelto yield better gradient gels.

What is claimed is:
 1. A method for making a gel comprising the stepsof: a. holding a solution in a reservoir; b. transferring the solutionto a dispensing tip; c. delivering the solution to a gel chamber with alongitudinal axis by moving the dispensing tip along the longitudinalaxis; and c. curing the delivered solution in the gel chamber to formthe gel.
 2. A method for making a gel, comprising the steps of: a.holding a plurality of solutions in a plurality of reservoirs; b.forming a fluid of at least one solution from the plurality ofsolutions; c. transferring the fluid to a dispensing tip; d. deliveringthe fluid to a gel chamber with a longitudinal axis by moving thedispensing tip along the longitudinal axis; and e. curing the deliveredfluid in the gel chamber to form the gel.
 3. The method of claim 2 ,wherein each solution of the plurality of solutions is a polyacrylamidesolution with a unique concentration.
 4. A method for making a gradientgel, comprising the steps of: a. holding a first solution at a firstreservoir and a second solution at a second reservoir; b. connecting thefirst reservoir and the second reservoir with a channel; c. connectingan outlet with the second reservoir, wherein the outlet communicateswith the channel so that a fluid of the first solution and the secondsolution is formed at the outlet; d. transferring the fluid to adispensing tip; e. delivering the fluid to a gel chamber with alongitudinal axis by moving the dispensing tip along the longitudinalaxis; and f. curing the delivered fluid in the gel chamber to form thegradient gel.
 5. The method of claim 4 , wherein the first solution is apolyacrylamide solution with a first concentration.
 6. The method ofclaim 5 , wherein the second concentration is no less than the firstconcentration.
 7. The method of claim 4 , wherein the second solution isa polyacrylamide solution with a second concentration.
 8. The method ofclaim 7 , wherein the second concentration is no less than the firstconcentration.
 9. A segmental linear gel made according to the method ofclaim 4 .
 10. A method for making a plurality of gels, comprising thesteps of: a. holding a plurality of solutions in a plurality ofreservoirs; b. forming a plurality of fluids, each fluid having at leastone solution from the plurality of solutions; c. transferring theplurality of the fluids to a plurality of dispensing tips, eachdispensing tip having one fluid therein; d. delivering the plurality ofthe fluids to a plurality of gel chambers positioned in parallel therebydefining a longitudinal axis by moving the dispensing tips along thelongitudinal axis, so that each of the plurality of the dispensing tipsdelivers one fluid therein in motion into one of the gel chambers; ande. curing the delivered fluids in the plurality of the gel chambers toform the plurality of the gels.
 11. The method of claim 10 , whereineach solution of the plurality of solutions is a polyacrylamide solutionwith a unique concentration.
 12. A segmental linear gel, comprising: a.a first segment comprising a linear polyacrylamide gradient of high tomedium polyacrylamide concentration; and b) a second segment comprisinga linear polyacrylamide gradient of medium to low polyacrylamideconcentration, wherein the second segment is on top of and in continuouscommunication with the first segment.